In theory, polymerase chain reaction (PCR) is potentially the most sensitive among all existing methods for rapid detection of microbes in an analyte (e.g. a food sample or a clinical specimen). In cases where detection of a broad range of pathogens is required and some are difficult to culture in vitro or require a long cultivation period, the value of PCR is especially appreciated. For example, by targeting a conserved gene such as the 16S rRNA gene for bacteria or the 18S for eukaryotic pathogens such as fungi, random screening or detection may be achieved using just one PCR reaction. However, in practice, using broad-range PCR to detect minute quantities of microbes is still fraught with many challenges. Some factors that limit the practical application of PCR in microbial detection include the inherent susceptibility of PCR to inhibitors, contamination, and experimental conditions. One of the most difficult and long-standing problems is that of endogenous contamination of PCR reagents.
Reagents for performing PCR, even those available commercially, are obtained from bacterial sources. As such, the reagents, in particular, the Taq DNA polymerases, unavoidably contain some level of DNA contamination from the source bacteria. The contaminating DNA usually include more than one strain or species that cannot be identified as Thermus aquaticus or Escherichia coli, yet, they bear close homology to the species of Pseudomonas fluorescens, Pseduomonas aeruginosa, Alcaligenes faecalis, or Azotobacter vinelandii, all of which are clinically important. As a result, conventional PCR often co-amplifies these contaminants with the target microbial DNA, generating an exceedingly high rate of false positives, thereby, rendering PCR assays so unreliable that they are precluded from clinical applications.
In the past 20 years, numerous attempts have been tried to solve this problem.
Some examples include UV irradiation, restriction endonuclease digestion, ultrafiltration, and pretreatment of reagents with DNase I (ref 20-23). Unfortunately, all previous attempts either failed to completely eliminate false positives due to inherent limitations on reaction conditions or could not achieve the required level of sensitivity due to concomitant inhibition of the PCR reaction. As a result, it is generally believed that broad-range PCR is not clinically relevant, leaving most researchers to pursue multiplex PCR or other non-PCR methods that are expensive, difficult to optimize and cumbersome to perform.
Therefore, there still exists an unmet need to overcome the challenges of utilizing PCR for clinical detection of microbes.